Process for treating desoxyribo-nucleic acid



United States Patent Office 3,176,006 Patented Mar. 30, 1965 3,176,006 PRUtJESS T QR TREATiNG DESUXYRLIBQ- NUCLEIC AQTD Rudoif Karl Zahn, Goitihergweg 2, Frankfurt am Main 65, Germany No Drawing. iFiied Aug. 6, 1963, er. No. 300,169 9 Claims. (Cl. 260--211.5)

The present invention is directed to a process for obtaining desoxyribonucleic acid.

The desoxyribonucleic acid which is present in biologlical material, particularly in the gonads and spermatozoa of many marine animals is exposed, during isolation and purification, to the danger of denaturation or cleavage of the twin macromolecule. Denaturing can be avoided by maintaining a pH value of 7.0; by the ion strength being high; and by complexing or combining heavy metals (e.g., with a complex ion).

In addition certain chemical substances and solvents which are deleterious must be excluded, e.g., formaldehyde and ethylene glycol. Breaking of the molecule is prevented by excluding or restricting mechanical shearing forces. However, most dangerous is the degradation due to desoxyribonucleases which are associated with the desoxyribonucleic acid in the biological material or are g introduced by microorganisms under non-sterile conditions or are introduced and/ or become activated by the solvent.

In order to split oif the desoxyribonucleic acid from t e nucleoprotein complex originally present in the bioal material, which contain the desoxyribonucleases,

d nts are used. These substantially denature the protei fraction without modifying th desoxyribonucleic acid. In addition they inactivate the existing desoXyribonucleases which become liberated. Unfortunately, however, it has been found that this is not generally and not completely the case. Those desoxyribonucleases that are contained, for example, in the gonads of sea urchins, holothuria, mussels and ascidia, remain so active even after treatment with detergents, that when kept at room temperature, the desoxyribonucleic acid is substantially degraded or decomposed after a few days. Only a very small part of the desoxyribonucleic acid can then still be precipitated in fibrous form with alcohol.

It is an object of the present invention to provide a process for treating desoxyribonucleic acid solutions containing desoxyribonucleases to remove and/ or to inactivate the desoxyribonucleases. It is also an object of this invention to provide desoxyribonucleic acid solutions in which the desoxyribonucleic acid is preserved in the active state.

As disclosed hereinbefore, the detergent wash of the desoxyribonucleic acid associated with the nucleo-protein complex does not always deactivate all the existing desoxyribonucleases. It has surprisingly been found that these residual activities of the desoxyribonucleases can be eliminated by heating simultaneously with the addition of the detergents, i.e., the detergent wash is carried out at elevated temperatures. The elevated emperatures utilized are above 40 C. and up to the temperature at which the desoxyribonucleic acid is structurally deformed, i.e., the temperature at which the quasi-crystalline structure collapses because of the melting of the hydrogen bonds in the untreated twin molecule. Generally, the maximum deactivating etfect is obtained when washing with the detergents at a temperature of about 10 C. below the structural deformation temperature.

The structural deformation temperatures for desoxyribonucleic acids obtained from different sources differ. These temperatures are generally above 75 C., e.g., about 84 C. (the structural deformation temperature of desoxyribonucleic acid obtained from Tyr. phages) to about 95 C. (the temperature of material obtained from M. phlei),

as more particularly pointed out in J. Marrnur, P. Doty: Nature (London) 183, p. 1427 (1959). The desoxyribonucleic acid (DNA) obtained from different sources varies as to the total of guanine plus cytosine content. Generally the higher this total content in the desoxyribonucleic acid, the higher is the structural deformation temperature, as pointed out in N. Sueoka, I. Marmur, P. Doty: Nature (London) 183, p. 1429 (l959). The salt content of the desoxyribonucleic acid solution also affects the structural deformation temperature. Other variable properties of the desoxyribonucleic acid obtained from different sources are disclosed in D. 0. Jordan, The Chemistry of Nucleic Acids, Butterworth (1960). All the references noted are hereby incorporated herein.

The useful detergents are the anionic surface active agents. These generally include soaps, sulfated amides, sulfated alcohols, sulfated others, petroleum sulfonates, sulfated carboxylic acids, sulfonated aromatic hydrocarbons, sulfonated aliphatic hydrocarbons, or phosphated compounds, etc. illustrative of these anionic surface active agents are the following: dodecyl phosphate, dodecyl metaphosphate, dodecyl sulfate, xylene-sulfonate, xylenephosphate, sorbitan-mono-oleate, sorbitan-di-oleate, nonyl-sulfate, and nonyl-phosphate. The following agents are preferred: sodium dodecyl sulfate, sodium Xylene sulphc-nate, dodecyl phosphate, xylene phosphate, nonyl-sulfate. The sodium, potassium and amornnium salts of these compounds are useful. The detergent containing washing solution should contain at least 0.2 g./l. of the detergent. Between 0.5 g./l. and 1 g./l. are preferred. IItOis nolt contemplated that the solution contain more than The washing solutions may also contain such auxiliary solvent materials as ethyl alcohol, buffer-ions at low ionic strength, and phenol, etc. The washing solutions as well as the DNA-containing solutions treated all have pH values of about 7 and preferably 7.2. They also preferably contain sequestering agents such as the sodium salts of ethylenediaminetetraacetic acid, the sodium salt of nitrilotriacetic acid, etc. In the usual washing procedure, the detergent containmg washing solution is heated to a sufficiently high temperature so that when it is poured into the desoxyribonueleic acid solution, the total of the resultant solution attains the desired temperature. When the desoxyribonucleic acid solution is at room temperature, it requires only simple experimentation and/or calculation to determine the volume and temperature of the detergent-containing washing solution necessary to bring the total of the two solutions when they are mixed, to the desired temperature. It is also possible to mix the detergent-containing washing solution with the desoxyribonucleic acid solution at room temperatures and quickly heat to the desired temperature. This second procedure is possible only with relatively small volumes of material. It becomes impracticable using larger volumes in view of the preferred short time period for treating at the elevated temperature.

The treatment period according to the invention is advantageously 1 minute at a temperature of 70-75 C. and the higher the temperature, the shorter is the treatment period. Generally speaking, it is sufficient to have reached a mixing temperature (in the mixer) of 72 C. for 1 minute with desoxyribonucleic acid from vertebrate animals and with many others. However, where the de soxyribonucleases are not inactivated in the one minute treatment period, treatment must be prolonged until it is all deactivated. This may take fifteen minutes or even longer when treating particularly resistant materials. The washing solution should be used in a volume ratio to the desoxyribonucleic acid solution of at least 0.5 to 3.0; and preferably between 1 to 1 and Z to 1.

The washing solutions are aqueous solutions.

ature.

As the desoxyribonucleases are eliminated in the first working up step of the desoxyribonucleic acid, the possibility is provided for the preservation of desoxyribonucleic acid containing materials in an active form. This is particularly important for the technical production of relatively large quantities or desoxyribonucleic acid, since the recovery and the working up of the biological starting material can now take place at dillerent intervals, as a function of time and space. For example, when preparing desoxyribonucleic acid from the gonads of marine animals, the preservation can be effected at the place Where the animals are caught (on ship or on the coast), while the working up can'be effected later after transport, inland at the laboratory or technical plant of an industrial concern. In addition, relatively small catches do not have to be immediately further processed, but can be stored and worked up economically in large batches. It has been possible for preserved batches to be kept under observation for a period up to two and a half years, without any detectable cleavage or modifications of the desoxyribonucleic acid occurring when it is stored under sterile conditions.

The term desoxyribonucleic acid solution is used to jdefine the aqueous baths containing the acid with or without associated nucleoprotein complex material including 'desoxyribonucleases. These baths may be true solutions and/ or suspensions. As disclosed hereinbefore, the acid from different sources has somewhat different compositions and characteristics as is known to the art. Similarly the twin molecule may contain various functional groups substituted thereon. In addition the acid may be present in the form of a functional derivative thereof. The term desoxyribonucleic acid as used, includes the substituted acid and derivatives, such as glucosylated DNA and the DNA-RNA complexes.

The following examples are illustrative of the invention:

Example] .Production of the solution A 1 part by'volume of a solution of 8 g. of tetrasodium salt of ethylenediaminetetraacetate in 100 ml. of water and 1 part by volume of a solution of 10 g. of sodium dodecyl sulphate in 100 ml. of 40% aqueous ethyl alcohol are mixed at room temperature and adjusted with K CO to a pH value of 7.0:L-0.2. 9 parts by volume of twice distilled water are added and heating takes place until all alcohol has evaporated and the boiling temperature of water has been reached. The operation takes place at temperatures from 96 to 101 C.

Example 2.-Prducti0n of the suspension of the material B containing desoxyribonucleic acid 2.5 parts by volume of material containing desoxyribonucleic acid obtained from spermatozoa, testes, tumor cells and the like with an ionic strength higher than 0.1 (possibly to be adjusted by adding salt) are mixed with 7.5 parts by volume of twice distilled water in a heat-resistant mixing apparatus. Temperatures from 15 to 30 C. are used.

Example 3.Preservati0n of the raw malarial To 10 parts by volume of the aqueous mixture B in the mixing apparatus, there are added 10.5 parts by volume of a boiling solution A after starting up the stirrer device. Only where it is absolutely necessary (e.g., for breaking down tissue) is the mixer to run for a period longer than is necessary for thoroughmixing (1-3 seconds), since a decomposition of the desoxyribonucleic acid can occur as a result of the shearing forces. With the volumetric ratio indicated, a temperature of about 65 C. is used. In those cases where desoxyribonucleases are particularly resistant to heat, it is necessary to heat to a higher temper- The suspension C obtained under sterile conditions may be kept at a temperature below the structural deformation temperature for minutes while stirring moderately. However, this last step can be omitted if .Cil

the preceding treatment has already resulted in complete destruction of the desoxyribonucleases.

Thereafter, under sterile Working conditions and under or in still warm condition (room temperature), extremely finely powdered, sterile sodium'chloride free from desoxyribonucleases is added to a final concentration of 2 m and finally sterile desoxyribonucleases-free 2 m NaCl solution until the specific viscosity as (measured at 25) has fallen to below 2.

1 is the viscosity of the solution and no is the viscosity of the solvent. The substance is thereafter cooled under sterile conditions to 0-4- and, after l5 hours, the suspension can be further worked up in .a manner known per se (cg, by centrifuging, protein denaturation with chloroform, intermediate precipitation with alcohol, removal of ribonucleic acid with ribonuclease, precipitation and drying).

Similar deactivation of desoxyribonucleases has been obtained, using sodium xylene sulfonate in place ofthe sodium dodecyl sulfate. Desoxyribonucleic acid solutions from othersources than those disclosed in Example 2 has been successfully treated following the procedure of Examples 141.

The preserved suspension can however also be stored under sterile conditions for a relatively long time at ()4- (3., possibly after adding antibacterial substances.

In the preparation of desoxyribonucleic acid, it is unavoidable that desoxyribonucleases united with the struc ture become free. The concern is to denature these suddenly, since the tissue desoxyribonucleascs can destroy desoxyribonucleic acid in seconds. However, in this operation, a denaturing of the desoxyribonucleic acid must be avoided. This is effected by the hot detergent solution being poured into the cold substratum (and under no circumstances in the converse manner). Pouring both solutions simultaneously in the right proportions together into one container or causing a common turbulent flow through a common container, is obviously the equivalent of pouring the hot detergent solution intothe cold DNA- containing solution.

As many embodiments of this invention may be made without departing from the spirit and scope thereof, it is to be understood that the invention includes all such modifications and variations as come within the scope of the appended claims.

What is claimed is:

1. Process for treating desoxyribonucleic acid solutions containing active desoxyribonucleases capable of degrading the desoxyribonucleic acid comprising pouring into said desoxyribonucleic acid solution a hot aqueous solution containing an anionic wetting agent and being at a temperature sufiiciently high so that when added to the desoxyribonucleic acid solution, the resulting temperature of the mixed solutions will be between 40 C. and t-e structural deformation temperature of the desoxyribonucleic acid.

2. The process of claim 1 in which the hot aqueous solution is stirred with the desoxyribonucleic acid solution for at least 1 minute.

3. The process for treating desoxyribonucleic acid solutions containing active desoxyribonucleases capable of degrading the desoxyribonucleic acid comprising pouring into said desoxyribonucleic acid solution a'hot aqueous wash solution containing between .2 g./l. and 10 g./l. of an anionic wetting agent, said wash solution being at a temperature sufllciently high so that when added to the desoxyribonucleic acid solution, the resulting mixed soluions will be between 75 C. and the structural deformation temperature of the desoxyribonucleic acidfihe ratio of said hot aqueous solution to the desoxyribonucleic acid solution being between 0.5 :l and 3:1.

4. The process of claim 3 wherein the pH of said mixed solutions is about 7.2.

5. The process for treating desoxyribonucleic acid solutions containing active desoxyribonucleases capable of degrading the desoxyribonucleic acid comprising pouring into said desoxyribonucleic acid solution a hot aqueous solution containing between 0.5 g./l. and 1 g./l. of an anionic wetting agent, said hot aqueous solution being at a temperature sufliciently high so that when added to the desoxyribonucleic acid solution, the resulting mixed solution will be between 75 C. and the structural deformation temperature of the desoxyribonucleic acid, the ratio of said wash solution to the desoxyribonucleic acid solution being between 0.521 and 3:1.

6. The process of claim 5 wherein the anionic wetting agent is sodium dodecyl sulfate.

7. The process of claim 5 wherein the anionic wetting agent is sodium Xylene sulfate.

8. The process of claim 5 wherein the ratio between the wash solution and the desoxyribonucleic acid solution is about 1:1.

9. Process for preserving desoxyribonucleic acid solutions comprising 10 perature, with stirring, for at least one minute.

References Cited by the Examiner UNITED STATES PATENTS 2,415,826 2/47 Laufer et a1 260-211.5 15 2,719,844 10/55 Dimroth et al 260-2115 2,738,333 3/56 Goldsmith 260--2l1.5

LEWIS GOTTS, Primary Examiner. 

1. PROCESS FOR TREATING DESOXYRIBONUCLEIC ACID SOLUTIONS CONTAINING ACTIVE DESOXYRIBONUCLEASES CAPABLE OF DEGRADING THE DESOXYRIBONUCLEIC ACID COMPRISING POURING INTO SAID DESOXYRIBONUCLEIC ACID SOLUTION A HOT AQUEOUS SOLUTION CONTAINING AN ANIONIC WETTING AGENT AND BEING AT A TEMPERATURE SUFFICIENTLY HIGH SO THAT WHEN ADDED TO THE DESOXYRIBONUCLEIC ACID SOLUTION, THE RESULTING TEMPERATURE OF THE MIXED SOLUTIONS WILL BE BETWEEN 40*C, AND THE STRUCTURAL DEFORMATION TEMPERATURE OF THE DESOXYRIBONUCLEIC ACID. 